Abstract
Background: Efficient gene electrotransfer can be achieved by means of combinations of microsecond pulses (pulses of 100 mu sec in duration) that permeabilize the cell membrane and of millisecond pulses (pulses of hundreds of milliseconds) that electrophoretically bring the DNA in contact to the cell. Contrary to the microsecond and millisecond pulses, nanosecond pulses (nanopulses, pulses of a duration of a few tens of nanoseconds) act on the inside of the cell. We explored whether nanopulses could affect the expression of electrotransferred genes, improving the overall efficacy of the non-viral, electric pulse-mediated, gene transfer. Methods: DNA coding for the luciferase was electrotransferred into cells in culture using classical procedures. Then cells were exposed or not to nanopulses (20 nsec in duration) and incubated for 24 or 48 hr. Measurements were performed using a luminometer, and results were expressed as pg of luciferase/ mu g of protein. Results: Preliminary results show an increase in the expression of the electrotransferred luciferase gene after exposure of the cells to a nanopulse(s). This increase seems to be dependent on several parameters that are still under investigation: the number of nanopulses, the amplitude (field strength) of the pulses, the delay between the gene electrotransferred and the nanopulses, the concentration of the DNA, the exposure system, the incubation medium, etc. Conclusions: An increase of three times of the luciferase gene expression can be achieved with the application of just a 1 nsec pulse of 60 kV/cm, 60 min after plasmid electrotransfer, in an electroporation cuvette with a 1 mm distance between the electrodes. No loss of viability is associated with exposure to these nanopulses. Thus cell manipulation by means of electric nanopulse delivery may efficiently increase the overall efficacy of gene electrotransfer.